PROJECT SUMMARY A primary objective of tuberculosis (TB) treatment is to prevent the emergence of drug-resistant disease. Standardized treatment regimens that include isoniazid, rifampin, pyrazinamide, and ethambutol are the backbone of the directly observed therapy-short course strategy for global TB control. The emergence of drug resistance during anti-TB therapy presents a major challenge to these efforts. Treatment of multidrug resistant TB requires the use of more toxic and less effective second-line drugs, and thus the prevention of emergence of drug resistance is a key aspect of TB elimination efforts. Our ability to study the development of drug resistance during M. tuberculosis infection is severely limited by insensitive tools to detect rare drug-resistant mutants in mixed populations. Conventional drug susceptibility testing using the agar proportion method defines phenotypic drug resistance at a threshold of 1% colony growth on drug-containing media. Traditionally, heteroresistant TB has been defined as between 1-99% colony growth on drug-containing media, with 100% defined as full resistance. Importantly, M. tuberculosis isolates display molecular heteroresistance when mutant M. tuberculosis DNA- conferring drug resistance- is simultaneously detected alongside wild-type (i.e. drug-susceptible) DNA. However, a major limitation of conventional polymerase chain reaction (PCR) methodology is the inability to amplify rare mutant sequences in a background of abundant wild-type DNA. Our long-term goal is to develop a new tool (?SuperSelective? PCR methods) to detect and monitor heteroresistant M. tuberculosis infections during the course of treatment. The objectives of this application are to develop and test SuperSelective PCR methods for the detection of rare mutations in genes encoding isoniazid or rifampin resistance, despite an abundant background of wild-type M. tuberculosis DNA. This work will directly lead to clinical studies of patient factors that contribute to the emergence of rare drug-resistant mutants during anti-TB treatment. With this new understanding, we can direct interventions towards preventing anti-TB drug resistance.